Signaling activities of amelogenin gene splice products.

描述（由申请人提供）：这是一份修订后的申请，旨在研究牙齿形态发生过程中淀粉原蛋白pre-mRNA剪接产物参与上皮-间质信号传导的潜力。特异性淀粉原蛋白mRNA剪接产物已被证明具有bmp样活性，可在体内植入异位部位诱导软骨/骨，并在培养的间充质细胞中诱导软骨/骨表型分子的表达。我们已经证明，两个特定的剪接产物，长度分别为73和59个氨基酸，通过调节转录因子的表达起作用。在培养过程中，它们对牙胚发育有不同的诱导作用。它们对牙本质缺损的修复和牙髓矿化也有不同的效果。虽然原淀粉原基因被认为只在牙上皮中表达，但原淀粉原基因产物已被证明存在于牙本质中，并且我们有原位杂交的证据表明它们可能直接在早期的前成牙细胞中产生。其他研究表明，淀粉原蛋白可能参与牙骨质的形成。基于这些数据，我们假设淀粉原蛋白基因小剪接产物多肽是转录调节因子，在牙齿发育的重要部分上皮-间质信号传导中起作用。本研究提出了三个具体目的：1)利用原位杂交技术确认成牙细胞中存在淀粉原蛋白mrna，利用免疫组化技术确定成牙细胞表达淀粉原蛋白基因信息的牙齿发育阶段，并显示成牙细胞中肽产物的外观；2)利用基因阵列和其他技术详细阐明在培养过程中，肽对指导牙齿发育过程中成牙髓细胞和成骨水泥细胞表型表达的影响；3)更全面地确定多肽与间充质细胞相互作用以改变其表型表达的机制。这些研究的逻辑延伸将是在目标2中检查肽在更一般的异型上皮-间充质重组实验中的使用，并在目标3中分析两种肽对骨诱导和牙齿发育中间充质细胞中bmp样活性和表型表达的综合影响。在这次修订中，提供了更多的实验细节。

DESCRIPTION (provided by applicant): This is a revised application to study the potential of amelogenin pre-mRNA splice products to participate in epithelial-mesenchymal signaling during tooth morphoigenesis. Specific amelogenin mRNA splice products have been shown to have BMP-like activities leading to cartilage/bone induction when implanted into ectopic sites in vivo, and to induce expression of cartilage/bone phenotypic molecules in mesenchymal cells in culture. We have shown that two specific splice products, 73 and 59 amino acids in length, respectively, act by regulating the expression of transcription factors. They have different inductive effects on the development of tooth germs in culture. They also yield different effects on the repair of dentin defects and pulp mineralization. Although the amelogenin genes have been thought to be expressed only in the dental epithelium, amelogenin gene products have been shown to be present in the mantle dentin and we have evidence from in situ hybridization that they may be produced directly in early pre-odontoblasts. Others have shown that amelogenins may be involved in induction of tooth cementum. On the basis of these data we have hypothesized that the amelogenin gene small splice product peptides are transcriptional regulating factors that have a role in the epithelial-mesenchymal signaling that is such a prominent part of tooth development. Three specific aims are proposed for this study: 1) To use in situ hybridization to confirm the presence of amelogenin mRNAs in odontoblasts, and immunohistochemistry to determine the tooth developmental stages at which odontoblasts express the amelogenin gene message, and show the appearance of the peptide products in the odontoblasts; 2)To clarify in detail, using gene arrays and other techniques the effects of the peptides on directing the expression of odontoblast and cementoblast phenotypes during tooth development, in culture; and 3) To more completely determine the mechanisms by which the peptides interact with mesenchymal cells in general to alter their phenotypic expression. Logical extensions of these studies would be to examine, under Aim 2, the use of the peptides in more general heterotypic epithelial-mesenchymal recombination experiments, and, under Aim 3) to analyze the combined effects of the two peptides on the BMP-like activity on the phenotypic expression in mesenchymal cells in bone induction and tooth development. In this revision, more experimental details have been provided.